Chitin Breakdown:The Bacterial Way

By Merrill Leffler

Each year, 100 billion tons of discarded crustacean shell sink through the world's oceans - more than a billion tons of this have been molted by copepods alone, the rest from shrimp, crabs and multitudes of other crustaceans. A major component of these shells is chitin - originally called animal cellulose - an insoluble polysaccharide that gives shells their flexible toughness.

"But if you sample marine sediments," says Saul Roseman, "you will find only traces of chitin - it's just not there." The reason: bacterial recycling. Bacteria chemically grind the shell away with a tool box full of proteins and enzymes, breaking down chitin's fibrous structure to simple sugars, which the microbes use as food.

Were it not for this recycling, says Roseman, a biochemist in the Department of Biology at The Johns Hopkins University, carbon and nitrogen in the chitin would simply accumulate, depleting the oceans in less than 50 years. "If chitin deposits weren't recycled," he adds, "the White Cliffs of Dover would consist of chitin instead of diatoms."

Most of these chitin-hungry microbes are from the genus Vibrio, ubiquitous motile aquatic forms which are the most abundant of all marine bacteria. For several years now, Roseman has been detailing the biochemical processes that bacteria employ in breaking down chitin - from first detection, to attach-ment, to the compounds they release and the enzymatic processes involved.

The findings in his lab, says Roseman, have been overturning a century of scientific dogma.

According to the dogma, chitin conversion is a two-step process that involves the production of a monosaccharide and a disaccharide. Roseman and the graduate and post-graduates in his lab - they originally included Charley Yu and Bonnie Basler and now include Nemat Keyhani, Alexi Fomenkov and Xibing Li - have found that that is not the case at all and have been uncovering what Roseman calls completely unexpected bacterial processes at every step - a virtual cascade.

Saul Roseman's lab is overturning a century of scientific dogma on the processes of bacterial recycling of chitin.

The first thing bacteria have to do is "find," or sense, the chitin. When a crustacean molts, it releases an enzyme, chitinase, to loosen the shell. This produces a small amount of oligosaccharide (a class of short-chain sugars, the building blocks of the chitin polymer) which the Vibrios can detect. Once the bacteria arrive, they attach to the surface of the shell. Charley Yu found that Vibrio furnisii are continually assaying the potential food value of material. "If they can synthesize protein [from it], they'll stick; if not, they'll go away," says Roseman. Then the bacteria begin to produce the enzymes needed to break down and digest the chitin.

His group has discovered that bacteria produce suites of these enzymes - outside the cell, between the cell walls and plasma membranes, and within the cell itself - and that those enzymes are involved in releasing a cascade of oligosaccharides of different molecular weights. So far they have identified a series of nine steps leading from chitin to a simple sugar (fructose-6-phosphate) and ammonia.

By identifying the genes that express each protein and enzyme, he intends to clone each of those genes in order to overexpress proteins in greater volume. In this way, each protein can be completely characterized with respect to its catalytic and other functions.

"While the scientific goal," Roseman says, "is to completely delineate the steps involved and how each step is genetically and metabolically regulated," his work could lead to major changes in the commercial production of oligosaccharides.

Chitin oligosaccharides, for example, are known to play an important role in plant disease resistance by "triggering" a plant's defense mechanisms against invasion by fungi (which have chitin in their cell walls). Also, symbiotic bacteria release chitin oligosaccharides to signal the formation of root nodules, sites for nitrogen-fixation in plants such as beans and clover. Chitin oligosaccharides may have potential use in human medicine as well. Recent studies have revealed that animals and humans produce chitinase-like proteins - though just what their role is remains open to question.

Exploring such possibilities, however, has been nearly impossible because the costs of obtaining pure oligosaccharides that are suitable for research are so prohibitive.

While bacteria produce the oligosaccharides with ease, thanks to their natural enzymes, replicating this process in the laboratory is environmentally problematic since those techniques require heavy use of acids and bases. The process is also time consuming and, as a consequence, very expensive. Roseman should know. More than 30 years ago, he developed chemical techniques for isolating saccharide compounds, a process that requires treatment after treatment to obtain high quality compounds. Those same techniques are still in commercial use today for manufacturing oligosaccharides from chitin.

Pure oligosaccharides can cost from $5 to $15 a milligram - one small experiment can cost many thousands of dollars.

As an example of the potential of the methods under development in Roseman's lab, Nemat Keyhani has already produced 25 gram quantities of the pure, crystalline disaccharide from chitin, which he is using in his own research. If the laboratory's progress continues, the group could provide opportunities for uses that medical and agricultural research have not even begun to think about. "Equally satisfying," says Roseman, "is that the source of these opportunities is a product of the seafood industry that is now going to waste."